CG920 Genomics
Lesson 4
Forward Genetics
Jan Hejátko
Functional Genomics and Proteomics of Plants,
Mendel Centre for Plant Genomics and Proteomics,
Central European Institute of Technology (CEITEC), Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.muni.cz
 Forward vs. reverse genetics
 Use of libraries of insertional mutants in forward genetics
 Searching in libraries of insertional mutants according to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
 Identification of the mutated locus
 plasmid rescue
 iPCR
 Use of libraries of point mutants in forward genetics
 Positional cloning
Outline
2
 Forward vs. reverse genetics
Outline
3
„Classical genetics“ approach
1. IDENTIFICATION OF PHENOTYPE
2. GENE MAPPING
3. GENE IDENTIFICATION
- position cloning
„Reverse genetics“ approach
1. ISOLATION OF SEQUENCE-SPECIFIC
MUTANT
2. IDENTIFICATION OF
PHENOTYPE
3. PROOF OF CAUSAL RELATIONSHIP
BETWEEN INSERTION AND
PHENOTYPE
RANDOM MUTAGENESIS
„Classical“ genetics versus „reverse genetics“
approaches in functional genomics
EMS
T-DNA
(retro)transposons
4
 Forward vs. reverse genetics
 Use of libraries of insertional mutants in forward genetics
 Searching in libraries of insertional mutants according to:
 anatomically or morphologically detectable phenotype
Outline
5
Insertional mutagenesis in
forward genetics approaches
 Use of insertional mutagenesis for study of
carcinogenesis
 Infection of EμMyc mice by MoMuLV retrovirus leads to lymphomas
formation, which arose due to activation of Pim kinases (40 % activation
of Pim1, 15 % activation of Pim2), molecular targets of these kinases
were unknown
Mikkers et al., Nature Gen (2002)
?
6
 Infection of EμMyc pim1 mutants by MoMuLV retrovirus leads to
lymphomas formation, which in 90 % contain insertion nearby (activation)
Pim2
 Use of insertional mutagenesis for study of
carcinogenesis
Mikkers et al., Nature Gen (2002)
?
Insertional mutagenesis in
forward genetics approaches
7
 Use of insertional mutagenesis for study of
carcinogenesis
 Infection of EμMyc double mutants pim1, pim2 by MoMuLV retrovirus leads to
lymphomas formation, which can be expected to activate either one of the
signalling partner of Pim proteins (Y), one of the proteins of Pim signalling pathway
(X) or to activate some of the related pathways leading to lymphomagenesis (Z).
Mikkers et al., Nature Gen (2002)
?
Insertional mutagenesis in
forward genetics approaches
8
 Cleavage of genomic DNA and ligation of
special linkers, so-called splincerettes
(increasing the specifity of amplification)
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
9
 First amplification using specific primers
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
10
 Second amplification using nested
primers (increasing the specifity)
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
11
 Sequencing and localization of regions
adjacent to provirus by searching in
annotated databases of mouse genome
 Isolation of genomic regions adjacent to the insertion site
of the provirus
Mikkers et al., Nature Gen (2002)
Devon et al., Nucl Acid Res (1994)
Insertional mutagenesis in
forward genetics approaches
In case of splincerette, the primer is of the same sequence as the top strand and therefore it is
unable to act as a primer until the complement of this strand has been synthesized (from the
insert-specific primer at the right-hand side).
12
 Forward vs. reverse genetics
 Use of libraries of insertional mutants in forward genetics
 Searching in libraries of insertional mutants according to:
 anatomically or morphologically detectable phenotype
 metabolic profile
Outline
13
 Mass and automated analysis of
metabolites (up to 25.000) by GC-MS
techniques in libraries of T-DNA mutants
 Metabolic profiling of plants
Metabolic profiling
14
 Mass and automated analysis of
metabolites (up to 25.000) by GC-MS
techniques in libraries of T-DNA mutants
 Identification of interesting (even
comercially interesting) mutants
 Metabolic profiling of plants
Metabolic profiling
15
Metabolic profiling
 Mass and automated analysis of
metabolites (up to 25.000) by GC-MS
techniques in libraries of T-DNA mutants
 Identification of interesting (even
comercially interesting) mutants
 Fast and easy isolation of genes through
identification of sequences affected by
T-DNA
 Metabolic profiling of plants
16
 Possibility to use special techniques, e.g. microdissection
 Metabolic profiling of plants
Metabolic profiling
17
 Forward vs. reverse genetics
 Use of libraries of insertional mutants in forward genetics
 Searching in libraries of insertional mutants according to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
Outline
18
 Analysis of expression profile (pattern) of the gene
and identification of mutants with altered pattern of
expression
 Identification of mutants with a change in the
expression profile
Expression profile
19
 Analysis of expression profile (pattern) of the gene
and identification of mutants with altered pattern of
expression
 Identification of mutants with a change in the
expression profile
 Possibility of partial automation (virtual digital
microscopy)
Expression profile
20
WT
Expression profile
21
 Forward vs. reverse genetics
 Use of libraries of insertional mutants in forward genetics
 Searching in libraries of insertional mutants according to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
 Identification of the mutated locus
 plasmid rescue
 iPCR
Outline
22
 Identification of chromosomal rearrangements responsible
for bushy phenotype of Arabidopsis
 Description of phenotype
Identification of mutated locus
23
Identification of mutant
 Crinkled leaves
 No trichomes on leaves and
stems
 Late senescence
 Bushy phenotype (branching
defective)
24
 Male sterility, defects in
stamen filament elongation
(A,B)
(compare with wild type C)
Identification of mutant
25
 Identification of chromosomal rearrangements responsible
for bushy phenotype of Arabidopsis
 Description of phenotype
 Identification of T-DNA mutated region
Identification of mutated locus
26
1. Identification of region of genomic DNA adjacent to the left
border using plasmid rescue
 Restriction digestion (EcoRI)
of mutant genomic DNA
 Religation and transformation of
E. coli
 Isolation of plasmid DNA from
positively selected clones
 Identified sequence was
identical to gene for NAD7
coded by mtDNA
Identification of mutated locus
27
2. Identification of region of genomic DNA adjacent to the right
border using inversion PCR (iPCR)
 Restriction digestion
(EcoRI) of mutant genomic
DNA
 Purification, religation and
PCR using T-DNA specific
primers
 Cloning and sequencing
 Identified sequence was
not homologous to any
sequences with known
function
Identification of mutated locus
28
 Identification of chromosomal rearrangements responsible
for bushy phenotype of Arabidopsis
 Description of phenotype
 Identification of T-DNA mutated region
 Localization of T-DNA insertion site in Arabidopsis genome
Identification of mutated locus
29
Searching in library IGF-BAC
 Genome library containing 10.752 clones with an average size of an
insert of 100 kb
 Bacterial clones arranged in the microtiter plates
 Library loaded onto nylon filters for hybridization with the
radiolabeled probe
30
I. Sequences adjacent to the left border of T-DNA
 28 positively hybridizing clones in total
 19 of them located on chromosome 2
 18 of them similar with mtDNA
II. Sequences adjacent to the right border of T-DNA
 6 positively hybridizing clones in total
 all of them located on chromosome 2
Mapping with IGF-BAC database
31
left border of T-DNASequences adjacent to right and
Localization of genomic T-DNA adjacent to both left
and right T-DNA borders on chromosome 2
 There was probably an inversion of almost entire
chromosome 2
32
 Forward vs. reverse genetics
 Use of libraries of insertional mutants in forward genetics
 Searching in libraries of insertional mutants according to:
 anatomically or morphologically detectable phenotype
 metabolic profile
 expression of genes of interest
 Identification of the mutated locus
 plasmid rescue
 iPCR
 Use of libraries of point mutants in forward genetics
 Positional cloning
Outline
33
 Positional cloning
 Principle: co-segregation analysis of segregating population (mostly of
offspring of backcrosses) with molecular markers
 RFLP (Restriction Fragment Length Polymorphism)
 RAPD (Randomly Amplified Polymorphic DNA)
 SSLP (Simple Sequence Length Polymorphism)
 Polymorphism of genome (PCR products) length, amplified using
specific primers
 Detection by Southern blot (PCR after digestion of the genomic DNA
and ligation of adapters)
 Polymorphism of length of randomly amplified genome segments,
using short 8-10bp primers
 CAPS (Cleaved Amplified Polymorphic Sequence)
 Restriction fragment length polymorphism, genome segments
amplified by PCR
Identification of mutated locus
34
m m
M M
Col Col
+
M
Col Ler
m
M
+
M
+
M
Ler Ler
M M
m m
Col Ler
M M
m m
Ler Ler
m +
M M
Col Ler
m
M
+
M
Ler Ler
+ +
M M
Col Col Col Ler
+
M
+
M
M M
m +
Col Ler
+ +
M M
♂
Ler Ler
♀
Col Col
mm
M M
Preparation of mapping population
M
m+
M M
Positional cloning
35
Recombinant analysis – determining the percentage of
recombination between mutation and molecular marker
r [%] = number of chromosomes of Col /
number of all the chromosomes × 100
marker I – linked
5 mutants
1/10×100 = 10%
marker II - no linkage
6 mutants
7/12×100 = 58%
• Analysis of approximately 2000 mutant lines
• Determining the closest (still) segregating marker
• Identification of mutation by sequencing
36
Map of DNA molecular markers
37
Markers for fine mapping
38